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Proteolysis-independent regulation of the transcription factor Met4 by a single Lys 48-linked ubiquitin chain

Nature Cell Biology volume 6, pages 634–641 (2004)Cite this article

Abstract

The ubiquitin ligase SCFMet30 is required for cell cycle progression in budding yeast. The critical function of SCFMet30 is inactivation of the transcriptional activator Met4. Here we show that a single ubiquitin chain is attached to Met4 through lysine at position 163. Inhibition of Met4 ubiquitination by mutating lysine to arginine at this position constitutively activates, but does not stabilize, Met4. This supports a proteolysis-independent role of Cdc34–SCFMet30-catalysed Met4 ubiquitination. Surprisingly, the ubiquitin chain attached to Met4 is linked through Lys 48 in ubiquitin, a ubiquitin chain structure that is usually required for substrate targeting to the 26S proteasome. These results suggest that Lys 48-linked ubiquitin chains can have a regulatory role independent of proteolysis.

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Figure 1: Lys 163 is the ubiquitination site on Met4.
Figure 2: The ubiquitin chain on Met4 is linked through Lys 48 on ubiquitin.
Figure 3: Met4 ubiquitination on Lys 163 is required for Met4 inactivation.
Figure 4: Ubiquitination on Lys 163 does not induce Met4 proteolysis.
Figure 5: Met4 activity is not regulated by Met4 protein abundance.

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References

  1. Hochstrasser, M. Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30, 405–439 (1996).

    Article  CAS  Google Scholar 

  2. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    Article  CAS  Google Scholar 

  3. Schnell, J.D. & Hicke, L. Non-traditional functions of ubiquitin and ubiquitin-binding proteins. J. Biol. Chem. 278, 35857–35860 (2003).

    Article  CAS  Google Scholar 

  4. Thrower, J.S., Hoffman, L., Rechsteiner, M. & Pickart, C.M. Recognition of the polyubiquitin proteolytic signal. EMBO J. 19, 94–102 (2000).

    Article  CAS  Google Scholar 

  5. Spence, J. et al. Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain. Cell 102, 67–76 (2000).

    Article  CAS  Google Scholar 

  6. Spence, J., Sadis, S., Haas, A.L. & Finley, D.A. Ubiquitin Mutant with Specific Defects in DNA repair and Multiubiquitination. Mol. Cell. Biol. 15, 1265–1273 (1995).

    Article  CAS  Google Scholar 

  7. Hofmann, R.M. & Pickart, C.M. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96, 645–653 (1999).

    Article  CAS  Google Scholar 

  8. Hoege, C., Pfander, B., Moldovan, G.L., Pyrowolakis, G. & Jentsch, S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002).

    Article  CAS  Google Scholar 

  9. Wang, C. et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346–351 (2001).

    Article  CAS  Google Scholar 

  10. Hicke, L. Protein regulation by monoubiquitin. Nature Rev. Mol. Cell Biol. 2, 195–201 (2001).

    Article  CAS  Google Scholar 

  11. Kaiser, P., Flick, K., Wittenberg, C. & Reed, S.I. Regulation of transcription by ubiquitination without proteolysis: Cdc34/SCF(Met30)-mediated inactivation of the transcription factor Met4. Cell 102, 303–314 (2000).

    Article  CAS  Google Scholar 

  12. Kuras, L. et al. Dual regulation of the met4 transcription factor by ubiquitin-dependent degradation and inhibition of promoter recruitment. Mol. Cell 10, 69–80 (2002).

    Article  CAS  Google Scholar 

  13. Thomas, D., Jacquemin, I. & Surdin-Kerjan, Y. MET4, a leucine zipper protein, and centromere-binding factor 1 are both required for transcriptional activation of sulfur metabolism in Saccharomyces cerevisiae. Mol. Cell. Biol. 12, 1719–1727 (1992).

    Article  CAS  Google Scholar 

  14. Thomas, D. & Surdin-Kerjan, Y. Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 61, 503–532 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Patton, E.E. et al. SCF(Met30)-mediated control of the transcriptional activator Met4 is required for the G(1)–S transition. EMBO J. 19, 1613–1624 (2000).

    Article  CAS  Google Scholar 

  16. MacCoss, M.J. et al. Shotgun identification of protein modifications from protein complexes and lens tissue. Proc. Natl Acad. Sci. USA 99, 7900–7905 (2002).

    Article  CAS  Google Scholar 

  17. Rouillon, A., Barbey, R., Patton, E.E., Tyers, M. & Thomas, D. Feedback-regulated degradation of the transcriptional activator Met4 is triggered by the SCF(Met30)complex. EMBO J. 19, 282–294 (2000).

    Article  CAS  Google Scholar 

  18. Skowyra, D., Craig, K.L., Tyers, M., Elledge, S.J. & Harper, J.W. F-Box Proteins are Receptors that Recruit Phosphorylated Substrates to the SCF Ubiquitin-Ligase Complex. Cell 91, 209–219 (1997).

    Article  CAS  Google Scholar 

  19. Skowyra, D. et al. Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1. Science 284, 662–665 (1999).

    Article  CAS  Google Scholar 

  20. Kornitzer, D., Raboy, B., Kulka, R.G. & Fink, G.R. Regulated degradation of the transcription factor Gcn4. EMBO J. 13, 6021–6030 (1994).

    Article  CAS  Google Scholar 

  21. Nakamura, S., Roth, J.A. & Mukhopadhyay, T. Multiple lysine mutations in the C-terminal domain of p53 interfere with MDM2-dependent protein degradation and ubiquitination. Mol. Cell. Biol. 20, 9391–9398 (2000).

    Article  CAS  Google Scholar 

  22. Petroski, M.D. & Deshaies, R.J. Context of multiubiquitin chain attachment influences the rate of Sic1 degradation. Mol. Cell 11, 1435–1444 (2003).

    Article  CAS  Google Scholar 

  23. Deffenbaugh, A.E. et al. Release of ubiquitin-charged Cdc34-S - Ub from the RING domain is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1. Cell 114, 611–622 (2003).

    Article  CAS  Google Scholar 

  24. Lee, C., Schwartz, M.P., Prakash, S., Iwakura, M. & Matouschek, A. ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. Mol. Cell 7, 627–637 (2001).

    Article  CAS  Google Scholar 

  25. Verma, R. & Deshaies, R.J. A proteasome howdunit: the case of the missing signal. Cell 101, 341–344 (2000).

    Article  CAS  Google Scholar 

  26. Elsasser, S. et al. Proteasome subunit Rpn1 binds ubiquitin-like protein domains. Nature Cell Biol. 4, 725–730 (2002).

    Article  CAS  Google Scholar 

  27. Muratani, M. & Tansey, W.P. How the ubiquitin-proteasome system controls transcription. Nature Rev. Mol. Cell Biol. 4, 192–201 (2003).

    Article  CAS  Google Scholar 

  28. Reed, S.I., Hadwiger, J.A. & Lorincz, A.T. Protein kinase activity associated with the product of the yeast cell division cycle gene CDC28. Proc. Natl Acad. Sci. USA 82, 4055–4059 (1985).

    Article  CAS  Google Scholar 

  29. Guthrie, C. & Fink, G.R. Guide to Yeast Genetics and Molecular Biology (Academic Press, San Diego, 1991).

    Google Scholar 

  30. Cherest, H. & Surdin-Kerjan, Y. Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: updating of the sulfur metabolism pathway. Genetics 130, 51–58 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Peng, J. et al. A proteomics approach to understanding protein ubiquitination. Nature Biotechnol. 21, 921–926 (2003).

    Article  CAS  Google Scholar 

  32. Gatlin, C.L., Kleemann, G.R., Hays, L.G., Link, A.J. & Yates, J.R., 3rd. Protein identification at the low femtomole level from silver-stained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry. Anal. Biochem. 263, 93–101 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to D. Finley, D Thomas, M. Tyers and V. Yu for reagents and yeast strains. We thank S. Reed, C. Wittenberg, M. Nomura and R. Steele for comments on the manuscript and C. Greer for support in setting up the lab. We also thank H. Zhang and J. Yen for plasmid constructs. K.F. was supported by the Austrian Academy of Science (APART-fellowship). This work was supported by a grant from the National Institutes of Health to P.K.

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  1. W. Hayes McDonald

    Present address: Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

Authors and Affiliations

  1. Department of Biological Chemistry, University of California Irvine, Irvine, 92697, CA, USA

    Karin Flick, Ikram Ouni, Chrissy Capati & Peter Kaiser

  2. Department of Cell Biology, The Scripps Research Institute, La Jolla, 92037, CA, USA

    James A. Wohlschlegel, W. Hayes McDonald & John R. Yates 3rd

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Correspondence to Peter Kaiser.

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Flick, K., Ouni, I., Wohlschlegel, J. et al. Proteolysis-independent regulation of the transcription factor Met4 by a single Lys 48-linked ubiquitin chain. Nat Cell Biol 6, 634–641 (2004). https://doi.org/10.1038/ncb1143

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